ABSTRACT Nearly all biological processes require proper production and degradation of cellular proteins. Maintaining this balance in protein homeostasis (proteostasis) is therefore essential to cellular fitness. Furthermore, a lapse in cellular proteostasis has been linked to the molecular basis of a wide variety of genetic diseases. Nevertheless, the manner by which the cell buffers adaptive swings in proteostasis and the impact of mutations on this process remains poorly understood. This is especially true concerning integral membrane proteins, which account for a quarter of the human proteome and the majority of current drug targets. Emerging evidence suggests the production of folded, functional, and properly localized membrane proteins in the cell is typically inefficient and sensitive to the effects of mutations. The interaction of nascent membrane proteins with molecular chaperones and other components of the cellular quality control (QC) network seems to play a central role in the efficiency of membrane protein biosynthesis and trafficking. However, the structural properties of co-translational folding intermediates as well as the nature of their interactions with molecular chaperones remain poorly understood. Nevertheless, the formation of these interactions implies that conformational defects are common among nascent membrane proteins. Based on the physicochemical mechanisms of cotranslational membrane protein folding, we hypothesize that the formation of non-native topomers during biosynthesis drives the QC-mediated retention of nascent proteins in the ER. Using the G-protein coupled receptor rhodopsin as a model system, we have employed a novel protein engineering approach to demonstrate that the activity of cellular QC is sensitive to the topological energetics. Moreover, we provide preliminary evidence that the pathogenic misfolding of rhodopsin, which is associated with retinitis pigmentosa, can arise from the stabilization of a non-native topomer. Using this approach, we will probe the nature of the interface between the topological energy landscape and the activity of the cellular QC network. To gain insights into the generality of these findings and the evolutionary trade-offs between folding and function, we will employ a novel adaptation of deep mutational scanning to survey the proteostatic effects of every possible point mutation in rhodopsin. The results will reveal whether the conformational equilibria of rhodopsin has evolved to be metastable or to maximize the efficiency of biosynthesis. Computational analysis of the results will also provide insights into the nature of the structural defects that give rise to proteostatic perturbations. Finally, we provide preliminary evidence that the constraints of cotranslational folding impose a contact order bias in the native structural ensembles of integral membrane proteins. To explore this paradigm, computational analyses of polytopic membrane proteins of known structure in conjunction with experimental measurements of helical interactions will be employed to determine the extent to which native, sequence-local contacts influence co-translational folding. Together, these results will provide fundamental insights into the mechanisms of membrane protein folding in the cell and the molecular basis of disease.